1. Field of the Invention
This invention relates to a method for producing tissue type plasminogen activator (hereinafter referred to as tPA), tPA which is produced and excreted by vascular endothelial cells and various tissue cells, lyses fibrin clots, namely thrombi. Thus, tPA is effective as a thrombolytic agent.
2. Description of the Prior Art
Various methods for producing tPA have already been suggested. An example of the representative methods is linking a DNA sequence of tPA to an appropriate promoter, recombining the sequence into an expression vector, transforming a host cell with the recombinant DNA sequence, cultivating the recombinant producing cell, thus transformed, in an appropriate medium to produce tPA, and then purifying the tPA using an affinity column and a gel-filtration column.
In such a method, examples of media for production of tPA generally include those containing inorganic acids, amino acids, vitamins, etc., such as Eagle Basal Medium (EMB), Eagle Minimum Essential Medium (EMEM), Dulbecco Modified Eagle Medium (DMEM) and RPMI 1640 medium, as basic components with supplementation of L-glutamic acid, 10% fetal calf serum (FCS) and sodium bicarbonate at a concentration of 1-2 g/liter to adjust the pH in the range 6.5-7.5.
For example, known methods include that in which Modified Eagle Medium supplemented with 10% fetal calf serum, sodium bicarbonate (1.2 g/liter) and an appropriate amount of L-glutamic acid is used for cultivation (Journal of Biological Chemistry Vol. 25, p. 7035) and that in whic cultivation is carried out in a basic medium such as DMEM, EMEM or PRMI 1640, supplemented with bicarbonate at a concentration less than 2.0 g/liter to adjust the pH in the range 6.5-7.5 (Japanese Patent Laid-Open No. 4233/1987).
According to the conventional methods described above, however, tPA production per unit cell number is extremely small and the productivity is poor so that the problems such as high production cost make them difficult to be applied for stable tPA production on a commercial scale.
On the other hand, it has been found recently that protein productivity by cells can be stimulated by increasing the osmotic pressure of the medium, although growth of the cells is slightly suppressed.
In general, in a process for producing a useful substance using animal cell cultivation, the conditions, such as osmotic pressure, pH and temperature, of the medium are controlled in extremely precise ranges. Particularly as to the osmotic pressure, it is controlled by modifying the composition of the medium in the range between 280 and 300 milliosmoles/liter which is the range equivalent to that of human blood and humor fluid or other body fluids.
In this regard, attempts have been made to improve productivity of a desired protein by increasing the osmotic pressure. For example, it is reported that in antibody production, the productivity of antibody producing cells is stimulated several times by increasing the amino acid concentration in the medium substantially to make the medium hypertonic up to 340 milliosmoles/liter (Japanese Patent Laid-Open No. 188062/1985).
However, the supplementation of amino acid to make the medium hypertonic may greatly influence cell metabolism, and in some cases the productivity of desired proteins may be adversely decreased.
The first object of the present invention is to provide a method in which the production of a desired protein, tPA, per unit cell number is improved. tPA has two molecular forms, single-chain tPA and double-chain tPA. Thrombolytic activity of double-chain tPA is higher than that of single-chain tPA. tPA has been conventionally developed as either the sole double-chain form or the mixture form of the double-chain and the single-chain form.
Double-chain tPA has high fibrinolytic activity, and it is highly possible that double-chain tPA activates plasminogen not in thrombi, where fibrinolytic effect is expected, but in the blood stream, which often causes clinical bleeding (Japanese Patent Laid-open Pub. No. 118717/1984).
However, single-chain tPA, which is considered to be a precursor of double-chain tPA, has high affinity to fibrin and is quickly converted to double-chain tPA once bound to fibrin.
Accordingly, single-chain tPA exhibits maximal plasminogen activity at clotting sites.
Thus, thrombolytic activity of single-chain tPA is relatively low and not exhibited in the blood stream. In consequence, for clinical use, single-chain tPA is in greater demand than double-chain tPA, and an effective method for the production of single-chain tPA is thus desired.
Known methods for preparing only single-chain tPA include a method in which cultivation and subsequent processing steps are carried out in the presence of aprotinin (European Patent Publication No. 41766), a method in which trypsin inhibitor or aprotinin is added in the culture medium for tPA-producing cells (Japanese Patent Laid-open Pub. No. 118717/1984), a method in which cultivation or induction production is carried out in a medium supplemented with aprotinin or benzamidine (Japanese Patent Laid-Open Pub. No. 19486/1986) and a method in which aprotinin or 6-aminocapronic acid is added in a purification process (Biochem. Biophys. Acta 719(2), 318-328, 1982). However, since aprotinin to be used is quite expensive, practical application on industrial scale is difficult. Consequently, a method in which low-molecular-weight chemicals are added in the place of expensive aprotinin derived from serum, namely, the methods in which antiplasmin agents such as epsilon-aminocapronic acid and tranexamic acid are added to the medium has been proposed (Japanese Patent Laid-Open Pub. No. 4233/1987).
However, significant promotion of the productivity of single-chain tPA is not accomplished by replacing aprotinin with low-molecular-weight antiplasmin agents or p-aminomethyl benzoic acid derivatives.
The second object of the present invention is to provide a method for improving the productivity of single-chain tPA to large extent.